人原发性胃恶性淋巴瘤裸小鼠原位移植模型的建立

Establishment of orthotopic transplantation model of human primary gastric malignant lymphoma in nude mice

目的为探讨胃恶性淋巴瘤的发病机制和治疗方法提供理想的动物模型。方法采用人胃恶性淋巴瘤术中原发灶和肝转移灶新鲜瘤组织植入裸小鼠胃黏膜下层，观察原位移植成瘤率，移植瘤的侵袭和转移率，进行形态学、染色体核型和流式细胞分析。结果13例人胃恶性淋巴瘤标本9例移植成功。依据WHO新分类标准，从中筛选出1株人胃原发性非霍奇金B细胞恶性淋巴瘤裸鼠原位移植肝转移模型HGBL-9903，1株人胃原发性非霍奇金B细胞恶性淋巴瘤裸鼠原位移植模型HGBL-9904和1株人胃原发性霍奇金B细胞恶性淋巴瘤裸鼠原位移植模型HGBL-9902。免疫组织化学示CD19、CD20、CD22、CD45和CD79a均阳性。染色体众数范围75～87条，流式细胞分析示DI值1.23～1.47，均为异倍体。3株模型分别传至83代、87代和89代，共移植裸鼠785只；自第3代起肿瘤的移植生长率和液氮冻存复苏成活率均为100％。HGBL-9903肝转移率为100％，脾转移率为36．7％，淋巴结转移率为57．6％。人胃恶性淋巴瘤在裸鼠胃壁内自主侵袭性生长，侵袭破坏胃壁各层组织结构。发生血液（肝，脾）转移，淋巴转移和腹腔内种植转移。结论HGBL-9904、HGBL-9903和HGBL-9902三株人胃原发性恶性淋巴瘤裸鼠原位移植模型，移植瘤的组织病理学、超微结构、DNA含量测定及染色体核型分析结果与来源人胃恶性淋巴瘤细胞相一致。完整的模拟了人胃恶性淋巴瘤患者的自然临床过程。

Objective To establish ideal animal models for exploring pathogenesis and experimental therapy of primary gastric malignant lymphoma. Methods Histologically fresh primary and liver metastatic lymphoma tissues removed from patients with primary gastric malignant lymphoma were transplanted into gastric submucosa of nude mice to observe tumorgenecity, invasion and metastasis for analysis of morphological characteristics, karyotype and flow cytometry. Results Successful transplantation of human primary gastric malignant lymphoma was seen in 9 out of 13 cases. According to the new classification standards of WHO on malignant lymphoma, there screened three strains of models, ie, orthotopic transplantation model （HGBL-9903） of human primary gastric malignant lymphoma （non-Hodgkin＇s B cell） in nude mice, metastasis model （HGBL-9904） of human primary gastric malignant lymphoma （non-Hodgkin＇s B cell） in nude mice and orthotopic transplantation model （HGBL-9902） of human primary gastric malignant lymphoma （Hodgkin＇s B cell） in nude mice. Immunohistochemistry showed positive CD19, CD20, CD22, CD45 and CD79a, with the number of chromosome for 75-87. Flow cytometry showed that DNA index （DI） ranged from 1.23 to 1.47, ie, heteroploid. Three models were passaged for 83, 87 and 89 generations respectively, where 785 nude mice were used for transplantation. Both the growth rate find resuscitation rate of liquid nitrogen cryopreservation of the transplanted tumors were 100 % from the third generation. In HGBL-9903, liver metastasis rate, spleen metastasis rate and lymph node metastasis rate were 100%, 36.7% and 57.6%, respectively . The transplanted tumors spontaneously grew in the stomach of nude mice to infiltrate and disrupt every layer of the gastric wall, with hematogenous metastasis （eg, liver and spleen involved）, lymph node metastasis and celiac planting metastasis occurred. Conclusions Three successfully established orthotopic transplantation models （HGBL-9904, HGBL-9903 and HGBL-9902） of human primary gastric malignant lymphoma in nude mice are consistent with the original human gastric malignant lymphoma in aspects of histopathological and ultrastructural features, DNA content and chromosomal karyotype and hence completely simulate the natural clinical process of human primary gastric malignant lymphoma.